Detection of RNA-RNA binding protein interaction assay using E. coli
Binding of heterologous RNA-BPs to target RNAs in E. coli inhibits translation of target genes. This method has been successfully applied to the study of a variety of RNA-BPs including HIV Rcv protein and nucleolin.
Operation method
An experiment to detect the interaction of RNA with RNA-binding proteins using the Escherichia coli system
Principle
Binding of heterologous RNA-BPs to target RNAs in E. coli inhibits translation of target genes. This method has been successfully applied to the study of a variety of RNA-BPs including HIV Rcv protein and nucleolin.
Materials and Instruments
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X-Gal reservoir IPTG storage solution lacZ buffer ONPG solution Sodium carbonate solution β-mercaptoethanol SDS Chloroform
Plate
1. Plasmids: pREV1, pLacZ-Rep, pACYC184.
2. strains: WMI and WMI/F'.
3. X-Gal reservoir: 30 mg/ml X-Gal dissolved in dimethylformamide and stored at -20℃.
4. IPTG stock solution: 1 mol/L IPTG dissolved in water and stored at 20℃.
5. lacZ buffer: 60 mmol/L Na2HPO4, 40 mmol/L NaH2PO4, 10 mmol/L KCl, 1 mmol/L MgSO4 (pH 7.0).
6. ONPG solution: 4 mg/ml ONPG was dissolved in water and stored at 4℃.
7. 1 mol/L sodium carbonate solution.
8. β-mercaptoethanol.
9. SDS.
10. Chloroform.
11. Detection indicator plate: LB-agar plate containing 100 μg/ml ampicillin, 35 μg/ml chloramphenicol, 60 μg/ml X-Gal, and/or IPTG.
12. Culture medium: LB culture medium containing 100 μg/ml ampicillin, 35 μg/ml chloramphenicol, and/or IPTG.
II. Methods of operation
1. Construction of plasmid pLacZ-Rep.
The RNA target sequence should be as close as possible to the SD sequence to maximize the transcriptional repression effect of RNA-BP binding to the target RNA. pLacZ-Rep plasmid contains two single restriction enzyme sites, KpnⅠ and HindⅢ, which can be used as insertion sites for the target RNA molecular sequence.
2. Construction of RNA-BP expression plasmid (pREV1 )
The plasmid pREV1 encodes HIV-1 Rev protein. There are two single restriction enzyme sites, ClaⅠ and NdeⅠ, upstream of the Rev gene, and SalⅠ and Bsu36Ⅰ downstream of the Rev gene; the Rev gene is excised by using these enzyme sites, and then it is replaced by the coding sequence of RNA-BP. In the process of plasmid cloning, we should pay attention to whether the reading frames match and make sure that the inserted RNA-BP coding sequence does not contain restriction enzyme recognition sites for cloning as far as possible; if it is impossible to avoid the above sites, other restriction enzymes can be used to replace them according to the situation.
3. Detection of transcriptional repression
The constructed reporter plasmid and RNA-BP expression plasmid were co-transformed into E. coli WM1/F', coated on X-Gal plate and incubated at 37℃ overnight. When β-galactosidase was expressed, the bacterial clones showed blue color, and the color of the clones was directly related to the expression level of β-galactosidase, so it was easy to distinguish the clones with double or more than double β-galactosidase activity. Comparison between bacterial clones co-transformed with the reporter plasmid and the RNA-BP expression plasmid and bacterial clones co-transformed with the reporter plasmid and the control plasmid, pACYC184, will allow us to determine whether the expression of lacZ is suppressed by the expression of RNA-BP. Clones with significantly reduced blue color indicate that RNA-BP expression triggers transcriptional repression.
The optimal conditions for RNA-BP expression need to be determined during the experiment, but it should be noted that high levels of some RNA-BPs may be toxic to the strain. The promoter of the RNA-BP-expressing plasmid pREV1 is regulated by the lac repressor, and therefore RNA-BP transcription is repressed in strains containing high levels of the lac repressor (e.g. WM1/F'). Moderate transcription of the RNA-BP gene can be obtained in these strains in the presence of the lac-inducing reagent IPTG.
(1) Observation of the transcriptional repression effect
The constructed reporter plasmid and RNA-BP expression plasmid were co-transformed into E. coli strain WM1/F', and E. coli strain WM1/F' was co-transformed with the reporter plasmid and the control plasmid, pACYC184, as a negative control.
② The transformed bacteria were divided into small portions and coated with LB-agar plates containing 0 μmol/L, 1 μmol/L, 2 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L, 50 μmol/L, or 100 μmol/L IPTG, and incubated at 37℃ overnight.
On the following day, compare the color difference between the different transformants and the control transformant (it is important to note that comparisons should be made between plates of the same IPTG concentration), and determine the lowest IPTG concentration of the assay indicator plate at which the color difference between the RNA-BP transformant and the control plasmid transformant is clearly visible.
(2) β-galactosidase activity assay
① Pick 4~6 clones from the lowest IPTG concentration plate obtained in the previous reaction that can clearly observe the color difference between the RNA-BP transformant and the control transformant into LB culture medium containing ampicillin, chloramphenicol and IPTG, and pick the control strain at the same time. shake at 37°C overnight.
② Inoculate 45 μl of overnight culture into 1.5 ml of LB culture medium containing ampicillin, chloramphenicol and IPTG, and shake at 37°C for 1.5~2.0 h ( OD600 is about 0.5).
③ Place the samples on ice for 20 min.
④ Pour the sample into a polystyrene tube and measure the OD600, and use the culture medium as a blank control.
⑤ Prepare several glass test tubes, add 0.5 ml lacZ buffer, 0.01% SDS and 50 mmol/L β-mercaptoethanol, two drops of chloroform in each tube, and then add 0.5 ml of bacterial culture medium in each tube. The culture medium was also used as a blank control. The reaction was carried out by vigorous shaking for 15 s and water bath at 28℃ for 5 min.
(6) Add 200 μl of ONPG solution to each tube and start the timer; the reaction is carried out in a water bath at 28°C until the liquid in the tubes turns yellow.
(vii) Add 0.5 ml of 1 mol/L sodium carbonate solution, shake to terminate the reaction, and record the reaction time of each tube. If there is no obvious color change in 3~5 h, terminate the reaction as well. Transfer the reacted sample to a microcentrifuge tube and centrifuge at 16000 g for 5 min. Transfer the supernatant to a polystyrene tube, taking care to avoid mixing with chloroform.
⑧ Read the OD420 and subtract the value from the blank culture medium tube.
⑨ β-galactosidase activity (miller unit ) = (2000 X OD420 )/(△t X OD600 ) for each bacterial culture sample, △t indicates the number of minutes of ONPG hydrolysis reaction.
The inhibitory activity of RNA-BP can be quantified assuming that there is minimal error during the experiment, that RNA-BP expression does not affect bacterial growth at all, that each individual clone grows well, and that there is little variation in β-galactosidase activity between samples. Therefore, at any IPTG concentration, if the average β-galactosidase activity in strains cotransformed with the reporter and control plasmids is X, and the average β-galactosidase activity in strains cotransformed with the reporter plasmid and the RNA-BP-expressing plasmid is Y, then the rate of inhibition, R, can be calculated (R=X/Y). The inhibition rate reflects the degree of inhibition of translation by RNA-RP binding to the target RNA in E. coli. In some experiments that will be described later, it is generally required that the inhibition of the reporter protein expression by RNA-BP should be 5-fold or more than 5-fold; if R≤5, the concentration of IPTG should be increased in order to express more RNA-BP. On the other hand, if the difference of β-galactosidase activity between the strains that express the RNA-BP is more obvious at a certain concentration of IPTG, it may be an indication that the expressed RNA-BP has a significant effect on the bacterial expression of the RNA-BP. On the other hand, if at a certain IPTG concentration, the difference in β-galactosidase activity between the strains expressing RNA-BP is obvious, it may indicate that the expressed RNA-BP is toxic to the bacteria, and then the IPTG concentration should be lowered.
Once the inhibitory effect of RNA-BP on the expression of the reporter gene is detected, it is necessary to further determine whether the inhibitory effect is caused by the specific binding of RNA-BP to the target RNA, and the simplest way to do so is to introduce a mutation in the coding sequence of the RNA-BP or in the target RNA, which affects the binding of the RNA-BP and the target RNA.
4. Application to the E. coli system
(1) Identification of mutations that can affect the binding between RNA-BP and target RNAs
After detecting the inhibitory effect of specific binding between RNA-BP and target RNA on reporter gene expression, this E. coli system can be used to screen for mutation sites in RNA-BP that affect the affinity of RNA-BP for target RNA. This screening method for mutants is based on the fact that the affinity of RNA-BP for the target RNA in E. coli is correlated with its inhibitory effect on translation. This method of mutant screening is based on the correlation between the affinity between RNA-BP and the target RNA and its inhibitory effect on translation in E. coli. Thus, in E. coli, RNA-BP mutants that increase the affinity between RNA-BP and target RNA inhibit lacZ synthesis more than natural RNA-BP, while RNA-BP mutants that decrease the affinity between RNA-BP and target RNA inhibit lacZ synthesis less than natural RNA-BP. By observing the color change of the transformed strains, it is possible to screen the RNA-BP mutant library for mutations that enhance or weaken the affinity between RMA-BP and the target RNA.
Genetic approaches can be altered to study the interactions between RNA-BP and its target RNA sequences, most commonly by mutating RNA-BP differently to study the effects of these mutations on RNA binding and reporter gene translational repression. Mutating RNA-BP across the board can help researchers determine the RNA biding surface of RNA-BP. If little is known about the binding between RNA-BP and the target RNA, the best approach is to randomly mutate RNA-BP and then screen for mutants that affect reporter gene lacZ synthesis, which can then be sequenced to determine which amino acid changes affect the binding between RNA-BP and the target RNA. Knowing which regions affect the binding between the two can narrow down the paradigm of mutations. Mutation frequency should also be controlled in experiments, and it is best to keep the overall mutation frequency at one or fewer mutations per gene. Too low a mutation frequency increases the screening effort required to screen for mutant strains affecting the interaction, while too high a mutation frequency complicates the analysis of the results.
When only a small region needs to be mutated, it is most convenient to synthesize a small oligonucleotide corresponding to the region to be mutated and then integrate it into the RNA-BP coding sequence. To facilitate this, the pREV1 plasmid contains an f1 phage replication initiation site that synthesizes single-stranded DNA, an intermediate in some of the oligonucleotide mutation processes. When mutating longer coding sequences greater than 50 or 100 bases, error prone PCR is currently the method of choice.
After the preparation of the mutant plasmid library, it can be directly transformed into strains that have been pre-transformed with the reporter plasmid; or it can be transformed into strains that have been transformed with the reporter plasmid after electrotransformation of the susceptible bacterium, and amplification within the bacterium. The transformed bacteria were coated with the detection indicator plate, and the strain co-transformed with the wild-type RNA-BP and the reporter plasmid was coated as the control, and the clones whose color was different from that of the control transformed strain were picked out after incubation at 37℃ overnight.
Although the affinity between RNA-BP and the target RNA was changed in many positive clones obtained by this method, the change in lacZ phenotype in some positive clones was not due to the change in the affinity between RNA-BP and the target RNA, but rather due to the change in the expression of RNA-BP in the transformed strain. A simple way to distinguish between these two causes of phenotypic changes in bacteria is to prepare protein lysates from logarithmically growing bacteria (including control strains co-transformed with wild-type RNA-BP and the reporter plasmid, and strains to be tested with altered phenotypes) and quantify RNA-BP expression by acrylamide gel electrophoresis, staining, or Western blotting, respectively, to screen out transformants with significantly altered RNA-BP expression. The transformants with significantly altered RNA-BP expression were screened out. Further studies could then focus on transformants with significant phenotypic changes but no significant changes in RNA-BP expression. When the expression level of RNA-BP cannot be observed directly, an indirect method can be used to determine the expression level of RNA-BP by the degree of inhibition of bacterial growth. The indirect method is to culture the control strain of wild-type RNA-BP co-transformed with the reporter plasmid and the test strain of mutant RNA-BP co-transformed with the reporter plasmid overnight in the culture medium without IPTG, inoculate the overnight cultured bacteria that have reached the growth plateau at the ratio of 1:100 (the concentration of IPTG in the medium slightly inhibited the expression of bison RNA-BP), and then shake at 37℃ for 2~3 h. Determine the OD600 of RNA-BP. ~The OD600 was measured at 37℃ for 2-3 h. The RNA-BP expression level in the mutant transformants with the same OD6000 value as the wild-type RNA-BP and the control strain co-transformed with the reported plasmid should be more consistent, and the transformants with far different OD600 values can be discarded.
After obtaining a series of phenotypically altered transformants, the results need to be further confirmed by β-galactosidase activity assay. Before further analysis, plasmid DNA needs to be extracted and purified, and then strains containing the reported plasmids need to be transformed, followed by the β-galactosidase activity assay. In this part of the experiment, the amino acids involved in the binding of RNA-BP to the target RNA can be found.
In addition, mutating the target RNA sequence in a similar way to mutating the RNA-BP coding sequence can also provide meaningful information for studying the interaction between RNA-BP and the target RNA. In this experiment, a library of mutated reporter plasmids was transferred into a strain containing wild-type RNA-BP, which was also subjected to phenotypic screening and β-galactosidase activity assay, and finally screened for nucleotide sequences in the target RNAs that affect their interactions with RNA-BP.
(2) Evaluation of binding affinity of RNA mutants or RNA-BP mutants
The E. coli system was established to express the RNA-BP at high levels and the lacZ reporter gene at low levels. A simple model of translational repression revealed that the intensity of flipper repression of RNA-BP in E. coli correlates with the affinity between RNA-BP and the target RNA. This hypothesis was confirmed by using purified proteins and RNAs to analyze the effect of dissociation constants on translational repression of different mutants of RNA-BP Rev and nucleolin with the target RNA. The researchers constructed a series of mutants of Rev and Riboprobe and tested the dissociation constants of the RNA-BP-target RNA complexes with the RNA gelshift RNA-binding assay, and the in vivo inhibition rate (R) of the same RNA-BP-target RNA complexes in the E. coli system was also examined. When the logarithm of the dissociation constant Kd was plotted against the logarithm of the repression rate (R-1), the mutants of both Rev and riboprobes yielded a straight line with good agreement (indicating a linear correlation), and this pattern for the RNA-BP Rev and riboprobes may be applicable to the other RNA-BPs as well. In addition, if a series of mutants were constructed, 6 to 8 of them were selected and purified and assayed for dissociation using an extracellular method, the RNA-BPs were assayed in vivo with the target RNA complexes. In addition, if after constructing a series of mutants, 6 to 8 mutants are selected and purified, and the dissociation constant Kd is detected by the extracellular method, and the inhibition rate R is detected by the intracellular method, and a standard curve is plotted; then if the inhibition of the other mutants is measured experimentally, then the standard curve can be used to deduce the dissociation constant Kd of this mutant.